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Washington University 
Doctoral Dissertations 



A Study of the Physiological 
Relations of Sclerotinia 
Cinerea (Bon.) Schroter 

B Y 

JACQUELIN S. COOLEY 




A Dissertation presented to the Faculty of Arts and 
Sciences in partial fulfilment of the requirements 
for the degree of Doctor of Philosophy, June, 1913 

Reprinted from Annals of the Missouri Botanical Garden, 
September. 1914, Vol. I. No. 3 



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A STUDY OF THE PHYSIOLOGICAL RELATIONS OF 
SCLEROTINIA CINEREA (BON.) SCHROTER 

J. S. COOLEY 

Formerly Rufus J. Lackland Fellow in the Henry Shaw School of Botany of 
Washington University 

Introduction 

This paper reports the results of an experimental study regard- 
ing certain pliysiological activities of the brown-rot fungus of 
stone fruits. Tlie investigation concerns itself primarily with 
the conditions influencing the penetration and infection of 
green and ripe fruits by the fungus in question, the action of 
the parasite on the host cell, and the secretion of the enzymes 
which act upon the cellulose and pectic substances of the host. 
The work was undertaken with the hope of throwing some further 
light upon the factors concerned in fungous parasitism. Our 
present conception of this subject is based upon fragmentary 
and, in some respects, contradictory evidence. However, each 
j'Car there are acquired new facts, or new applications of known 
facts, bearing upon this exceedingly involved and complex 
question. An examination into the history of investigations 
concerning the interaction of host and parasite shows that the 
study of this subject dates back to the work of the pioneers in 
plant pathology; modern methods and recent discoveries have, 
however, given an added impetus to research along this line. 

Progress in combating fungous diseases depends not only 
upon a familiarity with the life history of the parasite, but more 
especiallj' upon an intimate knowledge of the metabolism of the 
parasite and the nature of the changes which it induces in the 
host. Indeed, many of our recommendations for controlling 
parasitic diseases of plants will perhaps be modified when a 
more exact knowledge of the interrelations of host and parasite 
is gained. Furthermore, a more intimate knowledge of the 
physiological aspects of plant pathology will undoubtedly throw 
much light on the question of immunity and susceptibility. 

We should, of course, like to know more about the factors 
favoring or inhibiting parasitic action, as well as the conditions 

Ann. Mo. Bot. Gabd., Vol. 1, 1914 (291) 



292 ANNALS OF THE MISSOURI BOTANICAL GARDEN 

which influence the infection and the penetration of parasitic 
fungi. It would also be interesting to know why some fungi 
are so virulent and rapid in their destructive action on the host; 
for instance, it would be instructive to know whether it is due 
to the secretion of an enzyme, or a toxic substance (e. g., some 
acid), or to the disturbance of the osmotic relations of the host 
cells, or to some other perhaps unknown factor. For a study of 
some of these problems the writer has chosen as the organism 
Sclerotinia cinerea (Bon.) Schroter, the fungus causing the brown 
rot of stone fruits. This form is particularly suitable for the pur- 
pose since it is a virulent parasite, yet grows well as a saprophyte 
— readily lending itself to cultivation in the laboratory. 

Historical Review 

Space will permit only a brief review of some of the more 
important papers dealing with certain aspects of this subject. 
Much of the literature that is indirectly concerned with the 
problem, or that is fully reviewed or superseded by subsequent 
publications, will not be discussed here. 

In the period from 1858 to 1878 little experimental evidence 
appeared concerning the nature of the action of fungous para- 
sites, although several writers make mention of the penetration 
of host cells by fungous hyphae. Penetration was then fre- 
quently spoken of as merely a process of boring through ("durch- 
bohrung") the host tissue, Kiihn (34), as early as 1858, men- 
tioning this fact in a discussion of the potato-blight fungus. 
A few years later, in 1863, de Bary (1) speaks of the penetration 
of the host by Peronospora, and further makes mention of this 
fact in connection with his work on the rusts (2) ; again in his 
work 'Morphologie und Physiologie der Pilze, Flechten, und 
Myxomyceten' (3) he discusses the penetration of the host, but 
says he has no knowledge of the force that causes this boring 
into the host tissue. 

Hartig (26), in his early work on wood-destroying fungi, as 
well as in his later investigations, emphasizes the fact that fungi 
are able to destroy cellulose. By a microscopical study of 
diseased wood he found that the properties of the latter are 
very materially changed by the fungus; he did not, however, 
attempt to isolate an enzyme. 



1914] 

COOLEY — SCLEROTINIA CINERBA 293 

De Bary (4), in 1886, gives us the first important contribu- 
tion to our Ivnowledge concerning the action of parasites on host 
cells. This author, in his epoch-making research on the fungus 
now known as Sderotinia liberiiana, reports that the organism 
secretes a substance that discolors, plasmolyzes, and finally 
kills the host cells. This toxic secretion penetrates the host 
cells in advance of the fungus, killing them before they are 
actually pierced by the fungous filaments. De Bary was able 
to isolate this toxic substance, which he considered as probably 
an enzyme, and found that it would cause an injury to the 
host tissue similar to that produced by an attack of the fungus 
itself. He holds that the fungus will not grow on living tissues, 
for it attacks only through a wound and kills the cells in ad- 
vance of itself, thus not actually growing upon the living tissue. 
The product resulting from the disintegration of the cell wall 
of the host was thought to be a sugar that served as food for 
the fungus. In this connection de Bary also mentions finding 
oxalic acid encrusting the older fungous filaments. 

The next important paper on the interaction of host and 
parasite was that of Marshall Ward (51) published just two years 
after de Bary's work and concerning itself with a species of 
Botrytis causing a lily disease. In this excellent piece of work 
the author showed that the fungous hyphae on coming in 
contact with such solid substances as sections of a lily bulb, or 
even a cover glass, secrete from the tips drops of a substance that 
has a very peculiar effect on the host cell. He found that a 
water extract of this secretion when applied to sections of a 
lily bulb will cause the cell walls to swell and to assume an ab- 
normal appearance; the middle lamella is first dissolved and 
finally the entire cell wall is disorganized. Ward does not con- 
sider that this toxic secretion is stimulated by starvation. 

Several investigators have held that the penetration of 
many fungi is due to chemotropism, i. e., that penetration of 
the fungous hyphae is due to some stimulus which the constit- 
uents diffusing slowly from within the host cells exert. Biisgen 
(16), Miyoshi (39), Behrens (6), Schmidt (44), and others have 
adhered to the view that chemotropism is important, but more 
recent work, such as that of Fulton (25), does not uphold the 
theory. 



[Vol. 1 
294 ANNALS OF THE MISSOURI BOTANICAL GARDEN 

Behrens (6) investigated some of the physiological relations 
of saprophytes in comparison with parasites, using Mucor sto- 
lonifer, Penicillium sp., Botrytis cinerea, and Oidium { = Sclero- 
tinia^) fructigenum. This author holds that Sclerotinia does 
not produce a cellulose-dissolving enzyme, and that the fungus 
merely forces its way through the host tissue by a purely me- 
chanical force, or that, in some cases, it splits the middle lamella 
but does not dissolve it. In the case of the other fungi mentioned 
above he believes that an enzyme is secreted which dissolves 
the middle lamella. The cause of the injury due to Sclerotinia, 
he holds, is not that the cellulose walls or the pectin of the 
middle lamella is dissolved, but that the turgor and the osmotic 
relations of the penetrated cells are materially modified. Ac- 
cording to this author some substance diffuses through the 
walls and stimulates the fungus to bore through or between the 
cell walls. He demonstrated in Botrytis and Penicillium, more- 
over, a thermo-stable toxic body which disintegrated the host 
cells, and believes that these fungi secrete a pectin-dissolving 
enzyme which is different from that which acts upon cellulose. 

Nordhausen (40), at about the same time, made similar studies 
on Botrytis cinerea and comes to similar conclusions. He finds 
that the enzyme does not cause a strong swelling of either the 
middle lamella or the cellulose cell walls, the action in this respect 
being more like that of de Bary's Sclerotinia. Smith (46) stud- 
ied the parasitism of Botrytis cinerea, but in certain particulars 
did not get the same results as de Bary and Ward. Like them 
he finds that the parasite secretes some soluble substance that 
penetrates and kills the living cells in advance of the fungous 
filaments, but unlike Ward he could detect no swelling of the 
cell wall. Smith believes that this toxic substance is not an 
enzyme, for boiling does not inactivate it, but thinks that it is 
perhaps oxalic acid, since this substance is always present in the 
cultures and amounts in some cases to as much as two per cent. 
The analytical methods whereby the oxalic acid was determined, 
unfortunately, are not given. 

Schellenberg (43) investigated the action of several sapro- 
phytic and parasitic fungi on hemicelluloses from a number of 

' Wehmer, C. Ber. d. deut. bot. Ges. 16: 298-307. 1898; Saccardo, Syll. Fung. 
4: 34. 1886. 



1S14) 

COOLEY — SCLEROTINIA CINEREA 295 

different sources. He claims that these fungi act differently 
toward different celluloses, dissolving some and having no effect 
on others. The nature of the penetration and the action of 
certain parasites on the host tissue were also studied. There 
was no case in which Botrytis dissolved true cellulose, but it 
readily dissolved the hemicellulose part of the cell, leaving the 
cellulose intact. According to this author, therefore, the pene- 
tration and dissolving action of such parasites as Botrytis vul- 
garis is due to their ability to dissolve hemicelluloses. He 
considers that the middle lamella is largely composed of hemi- 
celluloses or closely allied substances. According to this view, 
therefore, organisms that dissolve the middle lamella are essen- 
tially hemicellulose-dissolving forms. As a result of his studies 
on Sclerotinia frudigena and S. cinerea, Schellenberg finds a 
different action on different fruits, but in no case does he report 
a splitting of the cells along the line of the middle lamella, as 
some previous investigators have reported. He believes that 
there is a slight dissolving action on that part of the cell wall 
which is in immediate contact with the fungous filament, but 
that the rest of the cell wall remains intact. In the twigs also 
he finds that the fungus dissolves the hemicellulose and leaves 
the true cellulose unacted upon. 

An extensive literature has developed concerning the enzymes 
of importance in the nutrition of fungi, but since these investi- 
gations either deal with saprophytes, or are only indirectly 
concerned with the work to be reported in this paper, it will be 
unnecessary to do more than mention some of the papers here. 
Among the more important contributors may be mentioned 
Ward (50, 52) , who was the first to use pure cultures of a wood- 
destroying fungus (Stereum), Biff en (9), who studied the biology 
of Bulgaria polymorpha, Bourquelot and H6rissey (13), who in- 
vestigated the enzymes in sporophores of Poly poms sulphureus, 
Czapek (18), who made his investigations with natural infections 
of MeruUus lacrymans and with other fungi, Kohnstamm (33), 
who worked on some species of MeruUus, Buller (14, 15), who in- 
vestigated sporophores of Polyporus squamosus, Van Iterson (28), 
who developed methods for isolating cellulose-dissolving bac- 
teria and fungi, and Dox (19), who investigated the enzyme 
action of species of Penicillium and Aspergillus. It is interest- 



[Vol. 1 
296 ANNALS OF THE MISSOURI BOTANICAL GARDEN 

ing to note that although we have every reason to beUeve that 
cytase is present in timber-decay organisms yet its presence has 
been demonstrated only indirectly by cytological methods. 
It is true, however, that many of the investigators mentioned 
above who found no cytase used the sporophores in their 
experiments and not the mycelium. 

The status of the subject of the enzymes concerned in the 
metabolism of parasitic fungi is given in Reed's recent publica- 
tion (42), which concerns itself with the enzymes produced by 
the parasitic fungus Glomerella ntfomaculans. This author has 
proved that the parasite produces many of the enzymes that 
had previously been reported for saprophytes, and by quan- 
titative methods has demonstrated different enzymes acting 
on the several classes of nutritive substances, such as carbo- 
hydrates, glucosides, fats, and proteins. He did not, however, 
investigate the cytolytic activity of the fungus but states that 
the nature of the diseased host would indicate that cytase very 
probably is not produced by this fungus. Peltier (41), as a re- 
sult of his investigations with Botrytis Fuckeliana, finds that 
the host cells are killed in advance of the fungous penetra- 
tion, and that the parasite secretes a thermo-stable toxic sub- 
stance, but, unlike Smith, finds no oxalic acid. The method 
of testing for oxalic acid unfortunately is not given. 

The action of bacteria on cellulose and other plant products 
has been extensively studied by a number of investigators, but 
for the purpose at hand it will suffice to cite some of the more 
recent publications in which the earlier literature is reviewed. 
The work of Jones (29, 30), which gives a good resume of the 
early work on this subject, is reviewed below under the dis- 
cussion of pectin. 

McBeth and Scales (38) report that a number of bacteria and 
fungi hydrolyze cellulose and claim that filamentous fungi play 
a very important role in the destruction of cellulose in soils. 
The cellulose-destroying fungi, according to these authors, act 
differently toward different kinds of cellulose, but their experi- 
ments do not seem to support this conclusion. Kellerman and 
McBeth (32) have also contributed to our knowledge of the cyto- 
lytic activity of fungi. Kellerman (31) has employed a method 



COOLEY — SCLEROTINIA CINEREA 297 

by which it is demonstrated that cytase diffuses in agar consid- 
erably beyond the region of hyphal penetration, and that a 
portion of the agar containing the enzyme dissolves cellulose in 
a manner similar to that of the fungus itself. 

The organism employed in my work was isolated from an 
infected plum twig, at Madison, Wisconsin. The original cul- 
tures were taken from a single colony in a Petri dish, this pro- 
cedure giving reasonable assurance that I was working with 
a single strain of the organism. Regarding the systematic 
relations of this organism a word may not be out of place here, 
since considerable confusion has arisen in the literature regarding 
the specific name of the organism causing the brown rot of 
stone fruits (27, 53, 37). Woronin (56) has made an important 
contribution designed to establish the systematic position of the 
two species Sclerotinia cinerea and S. frudigena. It has gener- 
ally been held that S. frudigena causes the brown rot of stone 
fruits in this country, while in Europe this fungus is found only 
on pome fruits; but Matheny (37) has recently given good evi- 
dence tending to show that it is S. cinerea which causes the 
brown rot of stone fruits both in this country and in Europe. 

Experimental Studies 

infection 

Some investigators, as, for instance, Zschokke (57), have held 
that Sderotinia cinerea is unable to penetrate sound fruit, while 
Smith (45), among others, has held that the fungus rapidly pene- 
trates and infects sound and unwounded fruit (peaches) . Casual 
observation in the field would seem to justify the former view, 
for those fruits in contact with other fruits or twigs, and there- 
fore liable to puncture or abrasion, are the ones that are usually 
found infected; indeed, field observations and laboratory exper- 
iments point to the conclusion that infection takes place much 
more readily, especially with immature fruits, when the cuticle 
is broken. One would, therefore, naturally raise the question 
as to whether or not infection can take place when the cuticle is 
unbroken, and if so under what conditions and in what stages 
of the development of the fruit. During the summer of 1913 



(Vol. 1 
298 ANNALS OF THE MISSOURI BOTANICAL GARDEN 

the writer performed a number of experiments which throw 
more light on the question of the infection of the host. 

Methods and Results. — The methods employed were as fol- 
lows: Plum twigs bearing leaves and fruit were broken off and 
brought into the laboratory, washed with a mercuric chloride 
solution (1-1000) and in sterile water. They were then sus- 
pended in sterile moist chambers prepared by placing moistened 
absorbent cotton in the bottom of wide-mouthed one-liter Erlen- 
meyer flasks that had previously been plugged and sterilized. 
Twigs having one or more green leaves were used in every case, 
for in this way green plums hang on the twigs and remain alive 
for some time. This method was especially applicable here, for 
it enabled one to maintain absolutely sterile conditions in a 
moist atmosphere and at the same time keep the host living 
and in a normal condition. The results of these infection ex- 
periments are given in table i. 

Discussion of Results. — From these results it is evident that 
plums were infected as early as June 27, at which time they were 
immature, in fact not more than half-grown. Infection did not 
take place when a spore suspension was placed on very green 
and immature plums unless the epidermis was broken or punc- 
tured. There were, however, some instances where plums re- 
mained healthy in the flask for two or three weeks and became 
infected only after the lapse of time had brought about an 
artificial maturity. On the other hand, plums that were ap- 
proaching maturity, though not mature, as well as mature fruits, 
may be infected by applying a spore suspension to the natural 
surface, i. e., a surface which has not been punctured or injured 
in any way. In this connection it should be mentioned that 
infection was much more readily accomplished when two plums 
were hanging so as to be in contact with each other than when 
they were not touching. This, no doubt, was due to the fact 
that a drop of water containing spores may be held between 
the plums long enough for spore germination and infection to 
take place. These results also indicate that infection takes 
place readily without puncturing when a portion of the mycelial 
felt is laid on the surface of either green or ripe fruit. 

It should be noted here that one can sometimes find plums in 
the field only half-grown which are affected with the brown-rot 



1911] 



COOLEY — SCLEROTINIA CINEREA 



299 



TABLE I 

RESULTS OF INFECTION EXPERIMENTS WITH SCLEROTINIA CINEREA 



Date 


Fruit 


Inoculating 
material 


Treatment 
of surface 


Method of 
inoculation 


Results 


June 27 


Green 
plums 


Spore 
suspension 


Cuticle killed 
by steam 


Surface 

appUcation 


+ +* 


June 27 


Green 
plums 


Spores 


Skin punctured 
with needle 


Needle 
puncture 


+ 


July 2 


Green 
plums 


Spores 


Skin punctured 
with needle 


Needle 
puncture 


+ + 


July 8 


Green 
plums 


Spores 


Skin punctured 
with needle 


Needle 
pimcture 


+ + 


July 8 


Green 
plums 


Spore 

suspension 


Untreated 


Surface 
application 


- 


July 8 


Sour 
cherries 


Spores 


Skin punctured 
with needle 


Needle 
puncture 


+ 


July 23 


Green 
plums 


Spores 


Skin punctured 
with needle 


Needle 
puncture 


+ + 


July 23 


Green 

plums 


Spore 

suspension 


Untreated 


Surface 

application 


- 


July 23 


Green 
plums 


Spore 

suspension 


Skin cut 


Surface 
apphcation 


+ + 


July 23 


Ripe 
plums 


Mycelium 


Untreated 


Surface , , 
application 


July 23 


Green 
plums 


Mycelium 


Untreated 


Surface 

application 


+ + 


July 30 


Green 
plums 


Mycelium 


Untreated 


Surface 

apphcation 


+ + 


Aug. 5 


Green 

plums 


Spore 

suspension 


Untreated 


Surface 

apphcation 


+ 


Aug. 13 


Nearly 
ripe 
plums 


Spore 

suspension 


Untreated 


Surface 
appUcation 


+ + 


Aug. 13 


Ripe 

plums 


Spore 

suspension 


Untreated 


Surface 

appUcation 


+ + 



* + + indicates that practicaUy every inoculated fruit became infected. 
+Lndicate8 that only a portion of the inoculated fruits became infected. 
— indicates that none of the inoculated fruits became infected. 



[Vol. 1 
300 ANNALS OF THE MISSOURI BOTANICAL GARDEN 

fungus, but SO far as the writer's observation indicates, infection 
in these cases takes place through the twig, or, in some cases, 
through another plum with which it is in contact and which in 
turn is infected through the twig. Nevertheless, field observa- 
tions also verify the laboratory work in that plums (especially 
certain varieties, such as Wood) when approaching maturity 
may be infected in the field without being in contact with other 
fruits and without having any visible punctures or wounds in 
the skin. All these experiments and observations point to the 
conclusion that penetration of the cuticle is a very important 
factor in the infection of fruits, especially immature fruits; that 
infection of very green fruits without punctures is rare; and, on 
the other hand, that maturing fruits without punctures may be 
readily infected both by spores and by a mycelial felt in the 
field and in the laboratory. 

PENETRATION 

The nature of penetration and the course of the hyphse of 
parasitic fungi in piercing host tissue is an interesting and im- 
portant question in connection with a study of the nature of 
parasitic action. In the case of the brown-rot fungus growing 
on the plum it is of importance to know whether or not the 
hyphiE merely follow the middle lamellae or whether they enter 
the cells wherever they come in contact with them. Previous 
investigators differ very widely in their opinions as to the nature 
and course of the penetration of the fungus in question, a condi- 
tion which is perhaps partly explained by the fact that different 
hosts were employed in the various investigations. Further- 
more, it appears that the methods employed in some of the 
researches were not of such a character as to readily yield com- 
plete information concerning all the facts in the case. 

In my own work a study of the penetration of the host tissue 
by the fungus was made by examining a number of sections of 
infected tissue in which the disease had reached various stages 
of development, and comparing them with sections of healthy 
tissue from the same fruit. For this purpose a special method 
was employed. 

Methods and Results. — Small pieces of fruit composed of 
diseased and sound tissue were cut from plums inoculated with 



1914] 

COOLEY — SCLEROTINIA CINEREA 301 

a pure culture of the fungus. These segments were immersed 
in 70 per cent alcohol just long enough to partially kill the 
fungous filaments and the host cells, yet not long enough to 
discolor the sound tissue or to modify or change the color of the 
diseased tissue in any way. From this material razor sections, 
containing both diseased and healthy tissue, were made, stained 
for a short time in eosin, and then partially destained with 
alcohol. If the pieces of plum had not remained in the alcohol 
for a sufficient length of time, the razor sections were immersed 
in 70 or 95 per cent alcohol before staining. By employing 
this method it is possible to stain the fungous filaments deeply, 
while the host tissue remains unaffected. Indeed, this method 
permits of a rather sharp color differentiation between the 
healthy and the diseased tissue, the latter being blackened by 
the disease. This method, though quite applicable for the pur- 
pose at hand, was primarily developed for another purpose, 
which will be discussed below. 

Since every fungous filament is very sharply differentiated, 
one may readily study the course of the hyphse with reference 
to the host cells. By staining, sectioning, and examining dis- 
eased material taken from the margin of the infected area, one 
finds the fungous hyphis penetrating the cells at any point of 
contact; indeed, after examining a number of specimens by the 
method reported above, the writer finds no indications that the 
fungous hyphse follow the middle lamella;, as has been reported 
by other investigators (57, 6) for pears and other fruits. 
The above method also enables one to contrast the cell walls 
of infected and penetrated cells with those of normal tissue. 
It is entirely possible that the fungous filaments, on coming in 
contact with a cell wall, secrete just enough enzyme to dissolve 
their way through the cell walls, leaving the walls of the host 
cells surrounding the hyphae entirely normal, i. e., without 
sweUing or disorganization. 

Another and somewhat different experiment was performed 
to get additional evidence on this point. From sound plums 
which had previously been rendered sterile by washing in bi- 
chloride of mercury solution (1-1000) and sterile distilled water, 
free-hand sections were cut with a razor sterilized in 50 per cent 
alcohol. The sections were arranged in hanging drop cultures 



[Vol. 1 
302 ANNALS OF THE MISSOURI BOTANICAL GARDEN 

and each inoculated with a drop of a very dilute spore suspen- 
sion containing two or three spores per drop. The progress 
of the fungus and the condition of the host cells were noted 
from day to day but no visible disintegration of the cell walls 
could be observed, nor did the fungus show any particular 
affinity for the middle lamellae. 

Conclusions. — We would conclude, therefore, as a result of 
direct observation on the host tissue, that the fungus penetrates 
the host very readily and rapidly, that it does not necessarily 
follow the middle lamellse in the plum and the peach, and that 
there is no visible general disintegrating action on the middle 
lamellae or on the cell walls of the living host. 

ACTION OF THE FUNGUS ON THE LIVING HOST CELLS 

A significant fact in the metabolism of the brown-rot fungus 
is that it induces such an exceedingly rapid decay in the infected 
fruits. This rapid decay might be connected both with a 
rapid growth of the fungus and with a pronounced power which 
the organism possesses of breaking down and changing the 
constituents of the host. Moreover, several representatives of 
the genus Sclerotinia have been reported to have the power of 
secreting an enzyme or some other substance which kills the 
host cells in advance of penetration. Were this the case, it 
would be expected that rapid decay would accompany the action 
of the parasite. Is this view applicable to the action of Sclero- 
tinia cinerea? The investigators who have made a study of 
this organism differ very widely in their views regarding the 
effect which it has on the host tissues, and it seemed desirable, 
therefore, to determine the relation of hyphal penetration to 
the death of the cells. 

Methods and Results. — In order to fix the material for this 
study, it was found satisfactory to proceed as follows : Small 
pieces of the host tissue were taken from the margin of the dis- 
eased area and placed in 95 per cent alcohol for a short time. 
Free-hand sections were made of this material so as to include 
both diseased and healthy cells, and the sections stained for a 
short time in eosin and subsequently decolorized in part with 
alcohol, if necessary to give the desired contrast. By this 



1914] 

COOLEY — SCLEROTINIA CINEREA 303 

method the fungus may be distinctly differentiated from the 
host tissue, the kiUing and staining agents having Uttle or no 
effect on the host cells. There is a more or less sharply differ- 
entiated line of demarcation between the injured and the sound 
cells, as indicated by the darker color of the former. The effect 
of the fungus is readily discerned by the blackening of the host 
tissue, this being especially noticeable in green plums. The 
discolored and poisoned cells are not at first plasmolyzed, and 
it is to be noted here that discoloration rather than plasmolysis 
should be taken as the index of the toxic action of this fungus 
on its host. It should perhaps be mentioned here, too, that the 
blackened cells shade off somewhat gradually into the hyaline 
healthy ones, and that, therefore, there is not always a sharp 
line of demarcation between the diseased and the healthy cells. 
However, in spite of these difficulties, I was convinced, after 
having examined a large number of sections of diseased and 
healthy tissue, that there is no positive evidence that the host 
cells are discolored, and therefore injured and poisoned, in 
advance of actual penetration by the fungus. 

The indirect method employed to determine the same point 
consisted in applying to sound fruits an extract from decayed 
plums. Fruits were disinfected with mercuric chloride solution, 
washed in sterile distilled water, and inoculated with Sclerotinia 
cinerea. When the plums had become thoroughly decayed 
the juice was extracted and filtered under sterile conditions 
through a Chamberlain filter. The juice thus obtained was 
incubated for one week at a temperature of 22-25° C, and also 
tested on nutrient agar plates, and found to be sterile by both 
methods. From sound plums, which had been disinfected in 
the usual manner, a cone-shaped plug was cut out and the 
resulting cavity filled with this sterile extract, — the controls 
being prepared in a similar manner, using sterile water instead 
of the plum extract. The results were negative, that is, the 
controls were not unhke those treated with the extract from 
decayed plums. 

The same experiment was repeated in a modified form by 
using thin razor sections of both green and ripe plums, the 
sections being made under sterile conditions as before, and ob- 
served in a hanging drop of sterile juice from decayed plums. 



304 ANNALS OF THE MISSOURI BOTANICAL GARDEN 

By means of this method one could readily observe any changes 
that might take place in the cells and make accurate compari- 
sons with controls. Frequent observations were made, and 
throughout this experiment, which continued for several days, 
one could not distinguish between the appearance of those 
sections in a drop of sterile water and those in the sterile extract 
from decayed plums. It is possible and perhaps probable that 
this fluid, being merely the juice of the fruit, was too dilute to 
be effective, but the experiment was made because of the 
possibility of positive evidence. 

Discussion of Results. — The initial stage in the injury caused 
by this fungus is shown by discoloration only and not by plas- 
molj^sis, and therefore one cannot draw conclusions with ab- 
solute certainty as to the poisoning effect of the extract on 
the cells of a cut surface, for the latter turn brown as soon as 
exposed to the air, just as when infected with the organism. 
It was comparatively easy, however, to observe that the extract 
had no effect on the cell walls, for no difference could be observed 
between the cell walls of the tissue thus treated and those of 
the control specimens. Even where the sections were left in 
the extract for several daj^s neither swelling nor disorganization 
of the cell walls or middle lamellae was noted. When sections 
of plum tissue were inoculated with one or more spores of the 
brown-rot fungus no cell-wall disintegration resulting from the 
growth of the fungus could be observed. A comparative study 
of sections of tissue, respectively exposed and not exposed to 
the action of the extract from decayed fruit, showed that no dif- 
ference could be detected between the two, and that, therefore, 
no enzyme with a perceptible cytolytic action exists under these 
conditions. It has been held by some, notably by Behrens (6), 
that the injury to the host cell is largely physical in that the 
fungus penetrates at such a prodigious rate that the fluids of the 
host cell are allowed to escape with loss of turgor to the latter; 
furthermore, that the osmotic equilibrium is soon destroyed, 
with plasmolysis and death ensuing. It is very probable that 
part of the rapid injury to the host can be explained on purely 
physical grounds, but this may not be the only factor involved, 
although we do not now know what chemical activity of the 
fungous cells may be concerned in the rapid kilUng of the host 
tissue. 



1S14] 

COOLEY — SCLEROTINIA CINEREA 305 

ACTION OF THE FUNGUS ON CELLULOSE 

A number of investigators hav'e regarded cellulose dissolution 
as a very important factor in the parasitism of many fungi; 
indeed, some of the earher workers seemed to consider this the 
prime factor involved. While it is a well known fact that there 
are many fungi, especially saprophytes, which hydrolyze, or dis- 
solve, certain celluloses, research extending over a wide field 
has revealed the nature of parasitism to be a very complex one 
in which other factors are as important as the dissolution of cel- 
lulose and the cell wall. 

It has been the writer's purpose to study from two different 
points of view the action of the brown-rot organism on celluloses, 
(1) by obseri-ing the action of the fungus on pure cellulose iso- 
lated from the host tissue, and (2) by studying microscopically 
its action on the host cell walls themselves. In the former 
study cellulose agar was used, the cellulose being isolated from 
plums by the methods discussed below. 

Methods and Results. — In the above mentioned study of the 
action of the fungus on pure cellulose, a variety of reagents, 
media, and methods for the preparation of cellulose were em- 
ployed, a brief account of which follows. Schweizer's reagent 
was prepared by adding a sUght excess (40 grams to the hter) 
of copper carbonate to dilute ammonium hydroxide solution 
composed of three parts of water to ten parts of ammonium 
hydroxide (sp. gr. 0.90). The copper solution was then shaken 
vigorously, allowed to stand over night, and the supernatant 
solution siphoned off. This is the procedure employed by Mc- 
Beth and Scales (38). 

Paper cellulose from filter paper was prepared according to 
the method given by McBeth and Scales (38) by dissolving 15 
grams of sheet filter paper in Schweizer's reagent, diluting about 
ten times with water, and precipitating the cellulose with a 
solution of one part of hydrochloric acid to five parts of water. 
This mixture was then further diluted to 15 or 20 liters, the 
supernatant Uquid siphoned off, and the residue washed re- 
peatedly with water until the precipitated cellulose was free 
from both copper and chlorine. After standing quietly for 
several days the clear liquid was siphoned off and the precipitate 
used for the preparation of cellulose agar. 



306 ANNALS OF THE MISSOURI BOTANICAL GARDEN 

Cellulose agar was made by adding about one per cent (esti- 
mated by the weight of the paper before treating with Schwei- 
zer's reagent) of precipitated paper cellulose, prepared as stated 
above, to a mineral nutrient solution, the complete medium 
having the following composition: 

Cellulose suspension 500 cc. 

Agar 10 grams. 

Monopotassium phosphate, 1 gram 

Magnesium sulphate, 1 gram 

Sodium chloride, 1 gram i ^qq ^^ 

Ammonium sulphate, 1 gram 

Calcium carbonate, 2 grams 

Tap water, 1000 cc. 

The insoluble precipitate appearing in the mineral nutrient 
solution was filtered off before the cellulose suspension and agar 
were added. Good results were also obtained by using 0.5 
gram of calcium nitrate instead of 2 grams of calcium carbonate, 
in which case filtering is unnecessary. The mineral nutrient 
solution having the composition tabulated above will be referred 
to as nutrient "A." 

Another nutrient solution very low in organic matter was 
also employed in the cellulose agar, but with rather unsatis- 
factory results. This solution, which will be referred to as 
nutrient "B," is that employed by Reed (42), and is made up 
as follows, the only organic material present being the small 
amount of sodium citrate: 

Ammonium nitrate 10 grams 

Dipotassium phosphate 5 grams 

Magnesium sulphate 1 gram 

Sodium citrate 1 gram 

Tap water 1000 cc. 

In making the cellulose agar this nutrient solution was used in 
exactly the same way as nutrient "A." 

Since previous investigators have held that the celluloses from 
various sources differ in their resistance to hydrolyzing enzymes, 
an attempt was made in this investigation to prepare a cellulose 
from a natural host — plums — of the parasite. In order to secure 
a cellulose that is modified as little as possible in the process 



COOLEY — SCLEROTINIA CINEREA 307 

of isolation three different methods were employed in preparing 
cellulose from plums, the resulting products being designated, 
for convenience in reference, respectively as soda cellulose, 
washed cellulose, and potassium chlorate cellulose. 

In the preparation of soda cellulose ripe plums were squeezed 
through cheese cloth and the pulp was washed thoroughly with 
water. The pulp was then treated with an 8 per cent solution 
of sodium hydroxide and heated in the autoclave at ten pounds 
pressure. After thoroughly washing the pulp with water the 
heating with alkali was repeated and the product given final 
washings until free from alkali. 

The second method of isolating cellulose — washed cellulose — 
consisted in washing the fruit pulp with water until free from 
substances soluble in cold water. Water was then added and 
the mixture heated in the autoclave at 15 pounds pressure, 
and washed. The operation was repeated as long as any water- 
soluble substances could be detected. This method, of course, 
gives an impure cellulose, yet the product is one that is free from 
water-soluble substances. 

The third method consisted in oxidizing, dissolving, and wash- 
ing out the plum pulp until a pure cellulose — potassium chlorate 
cellulose — was obtained. Pulp, secured from ripe plums in 
the manner stated above, was washed with cold water until 
the wash water was free from solutes, and then treated with a 
cold solution composed of 30 grams of potassium chlorate dis- 
solved in 520 cc. of cold nitric acid (sp. gr. 1.1). This mixture 
was kept in the ice box for about three weeks, at the end of 
which time the pulp was entirely white. This method' is said 
to yield a product that differs only very slightly from the original 
cellulose. 

The product obtained by these various methods was not al- 
lowed to dry, for it is possible that drying changes the nature 
of cellulose so that it is more resistant to the action of cytolytic 
enzymes. A part of the cellulose obtained by each of the pre- 
ceding methods was treated with Schweizer's reagent and pre- 
cipitated with hydrochloric acid and washed as stated above 
under the preparation of filter-paper cellulose. These three 
cellulose preparations thus treated with Schweizer's reagent, as 

' Fowler, G. J. Bacterial and enzymatic chemistry. 159. 1911. 



308 ANNALS OF THE MISSOURI BOTANICAL GARDEN 

well as the three corresponding untreated portions, were used 
in the preparation of cellulose agars, according to the method 
given above. The media were placed in test tubes of very small 
(8 mm.) diameter, and steriUzed. The tubes of melted agar 
were then cooled rapidly in cold water in order to bring about 
the hardening of the agar before the cellulose had had time to 
settle to the bottom of the tubes. 

Tubes of the various cellulose agars were inoculated with 
Sclerotinia cinerea and others with a species of Penicillium, 
which will be designated as P. expansurn^, isolated from decay- 
ing peaches and apples. Since these two fungi, viz., Sclerotinia 
cinerea and Penicillium expansum, act very differently toward 
the host, a word contrasting their action may not be out of 
place here. As a result of inoculating apples, peaches, or pears 
with a pure culture of Sclerotinia the host tissues are promptly 
killed, while the fruits remain practically as firm after complete 
decay as before inoculation. On the other hand, the fruits inocu- 
lated with the Penicillium become very soft and watery, develop- 
ing a pustule or sunken area where the infection took place. 
One may assume, therefore, that the Sclerotinia does not materi- 
ally affect the celluloses and pectic substances that make for the 
firmness of the fruit, while, on the other hand, Penicillium does 
affect these substances, causing the fruit to lose its firm consis- 
tency. Since these two fungi show such entirely different and 
opposing characteristics as regards their effect on the same 
host, it is interesting to compare their action in pure cultures on 
cellulose and pectin-like substances. Such a comparative study 
was made, the results of which are given in table ii. 

Discussion of Results. — The results given in table ii indicate 
that both Sclerotinia cinerea and Penicillium expansum exhib- 
ited in general a very slight hydrolytic action when grown on 
cellulose isolated from the plum, there being very slight action 
with both fungi on the soda cellulose and also on the potassium 
chlorate cellulose and no action on the washed plum cellulose. 
On the other hand, both fungi very readily dissolve filter-paper 

' A culture of this organism was sent to Dr. Chas. Thom, who very kindly examined 
it and gave as his opinion that it was P. expansum, or perhaps a strain of that species. 
The organism in question, when grown on the media employed by Thom, showed 
characters very similar to those of P. ex-pansum, as given by Thom (48). 



1614] 



COOLEY — SCLEROTINIA CINEKEA 



309 



TABLE II 

ACTION OF SCLEROTINIA CINEREA AND PENICILLIUM EXPANSUM ON CELLULOSE 





Nutrient 
solution 
added 


Sclerotinia cinerea 


Penicillium expansum 


Tj-pe of cellulose used 


Growth 


CeUulose 
hydrolysis 


Growth 


Cellulose 
hydrolysis 


Soda cellulose 


A 


+ +t 


-t 


+ + 


+ 


Soda cellulose 


B 


+ + 


- 






Potassium chlorate cellulose 


A 


+ + 


+ 


+ + 


+ 


Potassium chlorate cellulose 


B 


+ + 


+ 






Washed ligno-cellulose* 


A 


+ 


- 






Washed Ugno-cellulose* 


B 


- 


- 






Washed cellulose 


A 


+ 


- 


+ 


- 


Soda cellulose (Schweizer's) 


B 


+ 


- 


+ 


- 


Soda cellulose (Schweizer's) 


A 


+ + 


+ 






Washed cellulose (Schweizer's) 


A 


+ 


- 






Soda cellulose 


Peach 
juice 


+ + + 


- 


+ + + 


- 


Filter paper strips 


Peach 
juice 


+ + + 


- 


+ + + 


- 


Filter paper strips 


A 


+ + 


- 


+ + 


- 


Filter paper strips 


B 


+ 


- 


+ + 


- 


Filter paper strips 


0.5% 

glucose 

solution 


+ + + 


- 


+ + + 


- 


Filter-paper cellulose 


A 


+ + 


+ + + 


++ 1 +++ 



*Ligiio-cellulose is the name here given to cellulose from the vascular tissues of the 
plum, i. e., that part of the pulp which did not go through the cheese cloth. 

fGrowth and cellulose hydrolysis are indicated by +, the relative intensities of 
growth and degrees of hydrolysis being indicated by one or more + marks. Absence 
of growth and absence of hydrolysis are indicated by — . 



310 ANNALS OF THE MISSOURI BOTANICAL GARDEN 

cellulose, and, strange to say, Sclerotinia is just as active in this 
respect as PenicUlium-. In many cases the growth was as good 
on the plum cellulose as on the filter-paper cellulose, yet the 
hydrolytic action of the fungi was very much weaker on the 
former medium. No cellulose hydrolysis occurred where peach 
juice or some soluble carbohydrate, such as glucose, was added. 
It seemed probable at first that a very small amount of glucose, 
or peach juice, or sodium citrate would give the fungus a \agor- 
ous start and thus accelerate its cyto-hydrolytic activity, but 
the quantities of these substances employed was sufficient to 
exert a protective influence, there being a vigorous growth but 
no apparent cellulose hydrolysis. 

The fact that these fungi do not dissolve cellulose, derived 
either from the host or from paper, when other organic nutri- 
ents are supplied, verifies the writer's observation that Sclerotinia 
cinerea does not disintegrate the cell walls of the host tissues. 
Furthermore, the fact that the fungus dissolves paper cellulose 
very readily when it is the only carbohydrate supplied, leads 
one to conclude that the action of the fungus on paper cellu- 
lose in a nutrient solution low in carbohydrates is not neces- 
sarily a good criterion for judging the behavior of the fungus 
in the host tissue. In the host tissue there may be a form of 
cellulose different from that of paper, and it is furthermore 
very evident that there is present in the fruit an abundance of 
organic material evidently operating in a protective manner. 
The fungus fails to produce cytolytic enzymes when grown on 
plum or paper cellulose to which peach juice or even a very little 
sugar has been added, but acts vigorously on paper cellulose 
to which no organic nutrient has been added. It is rather 
peculiar that both fungi act much more readily on paper cellu- 
lose than on cellulose isolated from the fruits which are natural 
hosts for these organisms. 

Sclerotinia cinerea grows very slowly when first transferred 
to a nutrient medium poor in soluble carbohydrates, very few 
spores and no aerial mycelium being produced. At the expira- 
tion of a week or more one may observe that the fungous myce- 
lium has penetrated the surface layer of the agar, and at the 
expiration of two to three weeks, in case the fungus is growing 
on paper-cellulose agar, a clear translucent ring may be observed 



1914J 

COOLEY — SCLEROTINIA CINEREA 311 

in the agar just below the fungous filaments, thus indicating 
that the cellulose is being hydi-olyzcd. With increasing age of 
the fungus, this clear and almost transparent area gradually 
enlarges downward, although the fungus shows little or no cor- 
responding penetration. At the expiration of three weeks or a 
month, there is a very distinct, clear, and nearly transparent 
zone in the medium below the region occupied by the fungous 
mycelium. Since one could see very distinctly how far the 
fungous filaments had penetrated into the substrate, it was 
very evident that the cyto-hydrolytic enzyme had diffused 
beyond the limits of the mycelium. 

The method employed in this investigation for the demonstra- 
tion of cellulase was the same as that used by Kellerman in his 
recent work (31) and was utilized to demonstrate the fact that 
the cyto-hydrolytic enzyme secreted by this fungus penetrates 
the substrate considerably beyond the limits of the filaments 
themselves. Tubes containing cellulose agar, in which the 
fungus had been growing for four weeks, were disinfected exter- 
nally by washing with a bichloride of mercury solution, and cut 
off at a point about 12 mm. below the clear portion of the me- 
dium. The cotton plug was then flamed and pushed into the 
tube with a glass rod until the agar was partially shoved out 
of the cut end of the tube. The clear portion of the agar was 
then cut into disks about 12 mm. in thickness, which were laid 
on plates poured with nutrient cellulose agar, great care, of 
course, being exercised throughout the operation to maintain 
aseptic conditions. The plates so prepared were then placed 
in an incubator at 25°C. where they remained for two weeks, 
at the expiration of which time the cellulose was very distinctly 
hydrolyzed in a ring about the sterile slices of agar. Micro- 
scopic examination confirmed the macroscopic observation that 
these agar disks were free from any infection. 

As might be expected, the activity of the secretion of the 
enzyme cellulase is influenced by temperature, a fact which is 
well illustrated by the following experiment: Tubes containing 
cellulose agar inoculated with the brown-rot fungus were kept 
at temperatures of 10-12, 16-20, and 24-26°C. respectively, and 
at the end of twelve days the following results were noted: In 
the cultures maintained at 10-12°C. no apparent growth or 



312 ANNALS OF THE MISSOURI BOTANICAL GARDEN 

hydrolysis had taken place; those kept at 16-20°C. showed a 
good growth but no visible cellulose hydrolysis; and in those 
maintained at 24-26°C. there was about the same extent of 
growth as in the preceding series but accompanied by a very 
evident cellulose hydrolysis, a distinctly clear zone of dissolved 
cellulose surrounding the region occupied by the fungous myce- 
lium. It is therefore evident that even with approximately the 
same amount of growth cellulose hydrolysis is much more rapid 
at the higher temperature. 

An effort was made to determine whether or not it is possible 
to "train up" more active cyto-hydrolytic strains of the Sclero- 
tinia and Penicillium in question. On the one hand, these fungi 
were grown for several successive generations on peach-juice 
agar — a medium in which the organisms show no cytolytic ac- 
tivity. On the other hand, these fungi were cultivated for sev- 
eral successive generations on paper-cellulose agar — a medium 
which is low in soluble carbohydrates, and one in which the 
fungi exhibit considerable cytolytic activity. Tubes of paper- 
cellulose agar were then inoculated with the fungi grown in 
these two ways and careful observations were made to detect 
any differences in cyto-hydrolytic activity. No differences 
developed, however, from which it would appear that the source 
of cultures of Sclerotinia or of Penicillium does not materially 
affect the cellulose-dissolving capacity of these organisms, i. e., 
each fungus shows the same cellulose-hydrolyzing power wheth- 
er the organism was cytolytically active during the immedi- 
ately preceding generations or not. 

EFFECT OF THE FUNGUS ON PECTIC SUBSTANCES 

The power of organisms to change pectic substances has 
been considered an important factor in the disintegration and 
softening of host tissue by certain plant parasites. Before enter- 
ing into a discussion of the experimental phases of this subject, 
it will perhaps be well to give some idea of the present status of 
this question, as well as a very brief resum^ of the extensive 
literature which has accumulated about it. 

Fremey (23, 24), in 1840, was the first to report an enzyme act- 
ing on pectic substances. This enzyme, which he isolated and 
called pectase, induced the coagulation of pectin, Fremey attrib- 



COOLEY — SCLEROTINIA CINEREA 313 

uting this action of the enzyme to the presence of calcium salts. 
It is of interest to note that pectase was one of the first plant 
enzymes to be described. Bertrand and Mallevre (7, 8) con- 
cluded that pectose and pectase are almost universally present 
in green plants, being especially abundant in the leaves. These 
authors showed that acidity is an important factor in the inhi- 
bition of coagulation of pectic bodies by pectase, and also that 
either barium, calcium, or strontium is necessary for the action 
of pectase. 

Mangin (35, 3G), by microscopic tests, has thrown much 
light on the nature of the middle lamella and holds that pectose 
is very pronounced in the cell walls of young tissue. In the 
older cell walls, on the other hand, this author believes that 
calcium pectate predominates in the middle lamella, considering 
that the latter is largely if not entirely composed of this sub- 
stance and that it frequently collects on the surface of the cell 
walls adjoining intercellular spaces. Bourquelot (11), and 
Bourquelot and H^rissey (12) secured a thermo-labile enzyme 
from barley malt extract which acted upon a solution of pectin 
(taken from the gentian root), changing the latter in such a 
way that it was no longer coagulated by pectase. The action 
of this enzyme, which they called pectinase, was thought by 
them to be that of converting the pectin into reducing sugar. 
They also designated as pectinase an enzyme which dissolves the 
pectic coagulum (the latter has been supposed to be calcium 
pectate). A good resume of the status of the chemistry of 
pectic substances is given by Bigelow and others (10). 

A number of investigators have reported upon the action 
of bacteria on plant cells, including the effects of the organisms 
on the middle lamella. Winogradsky (55), Behrens (5), and 
others attributed the changes taking place in the flax plant dur- 
ing retting to the dissolving action which the bacteria exert on 
the middle lamella. It will be unnecessary to review here any 
more of the earlier work which has been done along this line, 
since it has been so thoroughly discussed in the comprehensive 
publications by Jones (29), and Jones, Harding, and Morse 
(30) on the soft rot of vegetables. These authors studied the 
effect of the soft-rot bacillus {Bacillus carotovorus) on the host 
and find that the organism is identical with what has been 



314 ANNALS OF THE MISSOURI BOTANICAL GARDEN 

designated as B. oleracece Harrison, and B. omnivorous Van Hall, 
and that it may possibly be identical also with Potter's B- 
destrudans. By many tests Jones has shown that this organism 
secretes an enzyme which causes the disintegration of the host 
cells by dissolving the middle lamella, which, according to the 
majority of investigators, is composed of salts of pectic acid. 
This author has further isolated from pure cultures of the or- 
ganism an extra-cellular enzyme, which he designated pectinase, 
that destroys the middle lamella of the cells just as does the 
growing organism. Jones, therefore, considers this enzyme 
responsible for the disintegrating action of the bacillus. 

In my own work I shall adopt the nomenclature used by Jones 
(29, 30) and Euler (21), namely, employing pectinase as the term 
to designate the enzyme inducing coagulation of a pectin solu- 
tion and also the hydrolysis of calcium pectate, or pectinate. 

Methods. — In order to determine the effect of the fungus on 
the middle lamella I have used two methods, (1) a microscopic 
study of the effect of the fungus on the host cells, and (2) a 
study of the effect of the organism on the substances (isolated 
from the host) which are commonly reported to be constituents 
of the middle lamella. The first method has been discussed 
above and may be dismissed here by stating that it yielded no 
positive evidence that the fungus dissolves the middle lamella. 
By the second method the problem was studied by isolating pec- 
tin from the host and studying the effect of the fungus on it 
and also on its salts, as, for instance, calcium pectinate. 

Pectin was isolated from plums by the following method: 
Thoroughly ripe fruits were steamed — no water being added, the 
juice filtered off and treated with Almen's reagent^ (to precipi- 
tate the protein) and with a very dilute solution of oxahc acid 
(to precipitate the calcium). It was found that under these 
conditions neither a calcium nor a protein precipitate was thrown 
down either by Almen's reagent or the oxalic acid, and this pro- 
cedure, therefore, was deemed unnecessary and was abandoned. 
The plum juice was carefully filtered through a Buchner filter 

'Abderhalden, E. Handbucli d. biochein. Arbeitsmethoden 2 : 391-92. 1910. 
Almen's tannic acid solution is made by treating 4 grams of tannic acid witli 8 cc. 
of a 25 per cent solution of acetic acid, and making up to 190 cc. with 40 or 50 per 
cent alcohol. 



1914] 

COOLEY — SCLEROTINIA CINEREA 315 

and the filtrate treated with 95 per cent alcohol until a floccu- 
lent coagulum of pectin was produced. This pectin was sepa- 
rated by means of a Buchner filter, redissolved in water, reprecip- 
itated with alcohol, again separated by means of a Buchner 
funnel, and finally dried at a temperature slightly higher than 
room temperature, — the reprecipitation being for the purpose 
of purification. It should be noted here that the plums were 
sufficiently acid to make the addition of hydrochloric acid to 
the alcohol unnecessary. 

Experiments with pectin and pectinase. — From the pectin 
isolated by the above method a saturated aqueous solution was 
prepared — some of the mineral nutrient solution' minus calcium 
being added, and the resulting solution rendered sterile by frac- 
tional sterilization. Test-tubes of this pectin solution were 
inoculated with Sclerotinia cinerea and Penicillium expansum 
with the result that both organisms produced a rather vigorous 
growth of mycelium and a few spores. At the expiration of 
one week the inoculated tubes showed a slight clear area just 
below the fungous felt due to the coagulation and settling out 
of the pectin in that part of the solution. The coagulation was 
at this time somewhat more pronounced in the Penicillium cul- 
tures than in those of Sclerotinia, yet very noticeable in both 
cases, beginning directly below the fungous felt and progressing 
toward the bottom of the tube. After two weeks the greater 
part of the pectin solution was coagulated, the flocculent coagu- 
lum, or precipitate, being very different from the precipitate 
produced in a pectin solution by a calcium salt. It should be 
emphasized here that every precaution was taken to maintain 
a calcium-free solution, and when it is considered that the addi- 
tion of calcium develops a reaction very different from that 
produced by the enzyme, and, furthermore, that the check gave 
no coagulation whatever, not even when allowed to stand a 
month or more, the conclusion would seem to be warranted that 
calcium is not necessary for the production of a gel by pectinase. 
Both Sclerotinia and Penicillium, therefore, produced a coag- 
ulum in an aqueous solution of pectin, while no such results 
were obtained in the controls, thus justifying the conclusion 

'Nutrient solution employed was the same as mineral nutrient solution A used in 
preparing cellulose agar, but without the calcium. 



[Vol. 1 
316 ANNALS OF THE MISSOURI BOTANICAL GARDEN 

that these two fungi are capable of producing pectinase. The 
cultures were kept at a temperature of 18-20°C. 

Experiments with calcium -pectinate. — Calcium pectinate was 
prepared by treating a water solution of pectin with freshly- 
made limewater (care being exercised to avoid an excess of lime), 
the product thus obtained being filtered off and thoroughly 
washed until it was no longer alkaline. The calcium pectinate 
thus prepared was used in making a pectinate agar in a manner 
similar to that employed in the preparation of cellulose agar, 
the same mineral nutrient solution (nutrient A) being used and 
the whole rendered sterile by fractional sterilization. After 
the last heating, care was taken to distribute the pectinate, 
which quickly settles to the bottom of the tubes, uniformly 
throughout the agar by stirring the medium with a sterile glass 
rod. These tubes were then inoculated with Sclerotinia and 
with Penicillium, the object being to compare the action 
toward pectic substances of two fungi that have entirely dif- 
ferent effects on the host cells, the former producing no soften- 
ing effects, while the latter causes a very rapid softening and 
disorganization of the host tissue. 

The inoculated tubes of pectinate agar prepared by the above 
method were kept at a temperature of 22-24°C. Contrary 
to expectations, there was very httle growth when no soluble 
carbohydrate was supplied, and, furthermore, no dissolving 
action on the calcium pectinate. On the other hand, when 0.5 
per cent glucose was added, both fungi produced a vigorous 
growth, but neither one gave any indication of pectinate hydroly- 
sis, or dissolution. Here again, as in the cellulose hydrolysis, 
the two fungi, Sclerotinia and Penicillium, behave alike. This 
is not in accordance with the observed behavior of these two 
organisms toward the host tissue. 

ACID RELATIONS OF THE FUNGUS 

Some investigators have held that the content of tannin (47) 
and of malic and other acids of the host determines whether or 
not the fungus can grow in the tissues and I'ot the fruit. In 
accordance with this view a fungus may not so readily attack 
green as ripe fruit, the former being supposed to exhibit a higher 



1914] 

COOLEY — SCLEROTINIA CINEREA 317 

content of these restraining agents. The question of the acid 
relation of the host tissue is one of fundamental significance 
and one that is worthy of considerable investigation; it is im- 
portant to know to what extent acidity may be a limiting factor 
in parasitism. 

A case in which a certain acid content is favorable for the 
fungus is developed by Falck (22). He finds the acidity of the 
substrate to be a conditioning factor for the growth of several 
species of Merulius. In this connection the author observes that 
Coniophora, in particular, acts to pave the way for Merulius in 
that the former organism renders the nutrient substrate deci- 
dedly acid, and thereby provides favorable conditions for the 
germination of the spores and the subsequent growth of myce- 
lium and fruit bodies of Merulius. In connection with the in- 
vestigation of the plum disease here discussed it would be well 
to know if the acidity of the fruit changes during the progress of 
its growth, and if so in what direction. It is also essential to 
know whether or not a change in the acidity of the host can 
account for the fact that ripe fruit is more susceptible to the 
disease than green fruit. Some experiments were planned, 
therefore, to determine to what extent the acidity of the host 
influences the attack of the parasite, and also to investigate 
what effects, if any, the fungus has with respect to the acid 
content of the host. 

In order to determine the changes in acidity which take place 
during the growth of the fruit (plums), several analyses for acid- 
ity were made at intervals during the summer. The plums for 
all of the analyses were taken from the same tree, a known 
weight of pulp being ground up in a mortar and squeezed 
through musUn. The acidity was reckoned in the number of 
cc. of N/10 NaOH required to neutralize one gram of plum 
pulp. The results were as follows: 

June 28, 1 gram plum pulp required 0.6G cc. N/10 NaOH for 
neutralization, 

Aug. 2, 1 gram plum pulp required 2.12 cc. N/10 NaOH for 
neutralization, 

Aug. 19, 1 gram plum pulp required 2.46 cc. N/10 NaOH for 
neutralization, 



318 



(Vol. 1 



ANNALS OF THE MISSOURI BOTANICAL GARDEN 



the fruit being market ripe on August 19. In these tests my 
results agree with those obtained by Bigelow and Gore (10) 
for peaches, and with those of Thompson and Whittier (49) 
for some other fruits. The last mentioned investigators, how- 
ever, found that the acidity of peaches decreases toward matu- 
rity. I have been unable to secure data covering the acidity of 
plums throughout the season. 

The above results show that the acid content of plums in- 
creases rather than diminishes toward the maturity of the fruit. 
The results of the experiments and field observations show that 
mature and ripe fruit is much more susceptible than the green 
and immature fruit. The above facts, showing that as the fruit 
approaches maturity the acidity increases while the suscepti- 
bility to the disease also increases, indicate that there is no 
close relationship between the low acid content of the host and 
susceptibility to the brown-rot fungus, and that we must look 
to other factors to explain infection as observed in the field. 
As pointed out, my experiments indicate that penetration is a 

TABLE III 

RELATION OF THE GROWTH OF SCLEROTINIA CINEREA TO THE REACTION OF 
THE MEDIUM 



Medium 


Acidity 


Growth after 
8 days 


Growth after 
16 days 


Spore 
production 


Cherry juice 


+2.3* 


-t 


+t 


+ 


Cherry juice 


+ 1.5 


+ + 


+ + 


+ + 


Cherry juice 


+ 1.0 


+ + + 


+ + 


+ + 


Cherry juice 


+0.15 


- 


+ 


+ 


Cherry juice 


-0.15 





+ + 





Cherry juice 


-0.30 





+ + 






*Aeidity is given in cc. of N/lO NaOH necessary to neutrahze 1 cc. of the juice. 

fXhe + sign indicates a fairly good mycehal gi'owth, or spore formation, and the 
— sign indicates that the growth was just perceptible; indicates no growth, or no 
spore formation. 



1014] 

COOLEY — SCLEROTINIA CINEBEA 319 

very important factor. It is possible that a study of the tannin 
content' might yield some relation of interest. 

A preliminary experiment was planned to determine the 
acidity at which the optimum growth and spore production of 
the fungus occurs. For this purpose the juice from ripe sour 
cherries was used. The juice was squeezed out of the cherries 
(no water being added) and a portion titrated to determine the 
acidity. Then 50 cc. of this liquid were put into each of a 
number of Erlenmeyer flasks of 125 cc. capacity; some of the 
flasks were left untreated, while others received various quanti- 
ties of N/ 10 NaOH to bring each to the desired acidity or alka- 
linity. The flasks were then sterilized and inoculated. The 
results are given in table iii. 

It is clear, therefore, that although the fungus eventually 
grows on a medium as acid as the natural juice of sour cherries, 
it grows more luxuriantly on a somewhat less acid medium. It 
is a rather significant fact that on the media near the neutral 
line the fungus at first shows no perceptible growth, but at the 
expiration of two weeks has produced nearly as much mycelial 
growth as on the acid medium. It is also of interest to note 
that we find spore formation abundant on the very acid media 
but entirely lacking on the alkaline media. This experiment 
indicates that the fungus can adjust itself to a slight degree of 
alkalinity. 

OXALIC ACID PRODUCTION BY THE FUNGUS 

The first important reference to oxalic acid production by 
fungi is in the publication by de Bary reviewed in a preceding 
section. He reports that the older hyphge of the fungus were 
encrusted with crystals of oxalic acid, and he attributed some 
of the poisonous action of the parasite to the production of this 
substance; in fact, he mentions oxalic acid fermentation. Since 
the appearance of de Bary's paper a limited number of investi- 

' Cook and Bassett and their associates (17) believe that there are enzjones in the 
host plant which may act upon cell constituents and play the role of alexins. They 
are of the opinion that tannin, as such, is not abundant in fruits, but that it may be 
formed by the action of oxidizing enzymes upon certain phenols. Injuries produced 
by parasitic fungi may accelerate the activity of the host in the production of tannin, 
the latter perhaps being toxic to the growth of parasitic fungi. 



320 ANNALS OF THE MISSOURI BOTANICAL GARDEN 

gators have reported the presence of oxahc acid resulting from 
the growth of both fungi and bacteria, but unfortunately much 
of this work is of little value, because methods of analysis are 
not given. The detection of this acid by some methods is very 
unsatisfactory. 

A few years after de Bary's work, Wehmer (54) published an 
extensive series of articles on this subject. He studied a number 
of fungi (mostly saprophytic) with reference to oxalic acid excre- 
tion, and of these he found Aspergillus to be the most active and 
Penicillium next, and, therefore, he confined his studies to these 
two fungi. Some of the factors concerned in the production of 
oxalic acid or its salts, according to Wehmer, may be summed 
up here: (1) A large yield of oxalic acid is not produced in the 
presence of free organic or inorganic acids, not being found in 
the medium when free acids exceeded 0.2-0.3 per cent, while, 
on the other hand, it can be formed in the presence of as much 
as 2-3 per cent of the salts of these acids. (2) The sources of 
nitrogen are very important, for the amount of the oxalic acid 
produced varies according to the kind and quantity of nitrog- 
enous compounds supplied. (3) Abundant oxalic acid forma- 
tion is favored by the addition of some basic phosphate, or at 
least some compound with which the acid can combine to form 
a soluble salt. (4) The effect of light or darkness on oxalic 
acid formation is inappreciable. (5) Temperature is an influ- 
encing factor in oxalate production, for the latter is inhibited by 
a high temperature, the temperature for a maximum oxalate 
production being, in fact, very near the minimum for the growth 
of the organism. 

Wehmer's analytical method consisted in precipitating out 
the oxalic acid, or its soluble oxalate, as the calcium salt, which 
was filtered off, dried to a constant weight, and weighed. Al- 
though this method is perhaps as well suited for this purpose 
as any other reported, it is open to criticism. A detailed dis- 
cussion, however, will not be given here. 

Wehmer holds that oxalic acid is a type of excretion, and that 
it is in some way connected with respiration, that is, with CO 2 
elimination. He considers that the variability in the amount of 
oxalic acid produced is due to its use in the metabolism of the 
fungus. Emmerling (20), in his contribution to this subject, 



1B14] 

COOLEY — SCLEROTINIA CINEREA 321 

emphasizes the influence of such nitrogenous substances as 
proteins, amino acids, and amides in the nutrient. He finds 
that Aspergillus niger when grown in non-amino acids, for 
example, tartaric, lactic, etc., produces no oxalic acid, whereas 
an abundant oxalic acid production results on such substances 
as peptone or aspartic acid. 

Smith (46) and Peltier (41) both conducted experiments to 
determine whether or not oxalic acid is present in media in 
which Botrytis has been growing. Peltier reported negative 
results, but Smith found oxalic acid and thinks that the pois- 
oning effect of the fungus is perhaps due to the presence of this 
acid. Unfortunately, neither of these authors gives his methods 
of analysis, and, with the exception of one incident in Smith's 
publication, the quantity of oxahc acid found is not reported. 
Peltier and others have been able to produce an injury with 
oxalic acid similar to that produced by certain parasitic fungi, 
such as Botrytis, yet this is not conclusive evidence that oxalic 
acid is the toxic substance secreted by the organism. 

The articles mentioned above constitute the chief publica- 
tions that have to deal with the production of oxalic acid by 
fungi. The publications on the production of oxalic acid by bac- 
teria and other plants will not be reviewed here. Whether ox- 
alic acid production is a phenomenon peculiar to certain genera 
or to certain species of the fungi, whether it is purely the result 
of external conditions, or whether it results primarily from cer- 
tain constituents of the medium, has not been clearly demon- 
strated. A series of experiments was planned in the hope of 
throwing some light on its production in the fungus here studied. 

The method of analysis employed was a modification of Weh- 
mer's method of precipitating the oxalate with calcium chloride 
and determining the amount of oxalate thus precipitated. 
This method, however, is not well adapted to the purpose at 
hand, especially when quantitative methods are used, and fruit 
juice is employed for the medium on which to grow the fungus. 
An attempt is being made to develop a method that will be 
better suited to our purpose. 

Culture media were prepared from peaches and plums by 
filtering the juices of these fruits through a Hill pressure filter 
under sterile conditions. The product thus obtained was 



(Vol. 1 
322 ANNALS OF THE MISSOURI BOTANICAL GARDEN 

placed in flasks and incubated for a week and found to be sterile, 
after which the flasks were inoculated with Sclerotinia cinerea. 
At the expiration of thirty-seven days these cultures were ana- 
lyzed and were found to contain the following amounts of oxahc 
acid per 50 cc. of the respective juices: 

Plum juice 0.0019 grams of oxalic acid, 

Peach juice 0.0077 grams of oxalic acid. 

Peach juice 0.0094 grams of oxalic acid, 

Control No trace of oxalic acid. 

Plum and peach juices that had been sterilized by heat, thereby 
precipitating some of the contained proteinaceous material, 
were also used as culture media, and here, too, every culture 
containing the fungus gave a positive test for oxalic acid. 

For investigating the production of oxalic acid by the fungus 
in the unaltered fruit, lots of 500 grams each of peaches were 
disinfected with bichloride of mercury solution, inoculated 
respectively with Sclerotinia, Penicillium, and Aspergillus niger, 
and kept under sterile conditions until the fruits were decayed, 
or, in the case of the Penicillium and AspergiUus, until partially 
decayed. The decayed fruits were then digested with hy- 
drochloric acid and analyzed for their oxalic acid content with 
the following results: 

Peach inoculated with Penicillium . . No trace of oxalic acid. 
Peach inoculated with Aspergillus . . .No trace of oxalic acid. 

Peach inoculated with Sclerotinia cinerea 

0.0087 grams of oxalic acid, 
Peach control No trace of oxalic acid. 

The results of these experiments with oxalic acid show that 
Sclerotinia cinerea when grown either on fruit juices or on 
peaches produces more or less oxalic acid as a result of its meta- 
bolism. It is also significant that the other two fungi employed, 
namely, Aspergillus and Penicillium, which are not natural 
parasites on the plum or the peach, produced no oxalic acid 
under the conditions in which the experiments were carried out. 

Summary 
1. The brown-rot organism will infect fruits which are im- 
mature, even penetrating those which are not more than half- 
grown or those in which the pits are still soft, provided the 



1914] 

COOLEY — SCLEUOTINIA CINEREA 323 

skin is punctured. Infection of green fruits is also effected when 
a portion of the mycelial felt of the fungus is laid on the surface 
of the plum. On the other hand, ripe or nearly mature fruits 
may be readily inoculated by sowing a spore suspension on the 
unpunctured surface. 

2. The fungus does not show any particular affinity for the 
middle lamella, but penetrates and permeates with equal avidity 
any part of the host tissue. 

3. A study of the effect of the organism on the host gives no 
positive evidence that a toxic substance is abundantly secreted 
in advance of penetration. 

4. The fungus shows very slight cytolytic action with respect 
to cellulose isolated from the plum, while, on the other hand, the 
organism readily hydrolyzes cellulose from filter paper when 
this is the only carbohydrate supplied. No general cytolytic 
action of the organism on the cell wall of the host is perceptible. 

5. An aqueous solution of pectin isolated from plums was co- 
agulated by Sclerotinia, thus indicating the secretion of the 
enzyme pectinase. In respect to its action on pectic substances, 
Sclerotinia cinerea behaves in a manner similar to that of Pen- 
icillium expansum, yet these two organisms produce very dif- 
ferent effects on the host, the former producing a firm rot and 
the latter a soft one. Neither organism will dissolve calcium 
pectinate. 

6. The experiments on the acid relations of the fungus indi- 
cate that the changing acidity of the host as the fruit reaches 
maturity does not explain the fact that ripe fruit is more sus- 
ceptible to the disease than green fruit. 

7. The brown-rot fungus produces oxalic acid when grown 
either on a fruit juice medium or on peaches. 

The writer takes pleasure in acknowledging his indebtedness 
to Professor B. M. Duggar for his advice and helpful criticism 
in this investigation. Part of this work was done during the 
summer of 1913 in the Laboratory of Plant Pathology of the 
University of Wisconsin, and the writer wishes to express his 
gratitude to Professor L. R. Jones for the courtesy extended to 
him while at Madison. 

Graduate Laboratory, Missouri Botanical Garden. 



324 annals of the missouei botanical garden 

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Washington University 

David F. Houston, • A.M., LL.D., Chancellor 
Frederic A. Hall, A.M., Litt.D., L.H.D.. LL.D., Acting Chancellor 

L The Department of Arts and Sciences (Skinker road and Lindcii boulevard) 

A The College 

George O. James, Ph.D., Dean 

B The School of Engineering 

Alexander S. Langsdorf, M.M.E., Dean 

C The School of Architecture 

Alexander S. Langsdorf, M.M.E., Dean 
John B. Robinson, Professor in Charge 

II. The Henry Shaw School of Botany (Shenandoah and Tower Grove avenues) 

George T. Moore, Ph. D., Engelmann Professor of 
Botany 

III. The Law School (Skinker road and Lindell boulevard) 

William S. Curtis, LL.D., Dean 

IV. The Medical School (Kingshlghway and Euclid avenue) 

Eugene L. Opie, M.D., Dean 

V. The Dental School (Twenty-ninth and Locust streets) 

John H. Kennerly, M.D., D.D.S., Dean 

VI. The School of Fine Arts (SUinker road and Lindell boulevard) 

Edmund H. Wuerpel, Director 

VII. The School of Social Economy (2221 Locust street) 
George B. Mangold, Ph.D., Director 



The following schools, each with its separate and distinct corps 
of instructors, are also conducted under the charter of the 
University: 

1 Smith Academy — for boys (Von Versen avenue and Windermere way) 

Frank Hamsher, A.B., Principal 

2 Manual Training School for boys (Von Versen avenue and Windermere way 

William R. Vickroy, Ph.B., Principal 

3 Mary Institute for girls (Waterman and Lake avenues) 

Edmund H. Sears, A.M., Principal 
On leave of absence. 



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